1. Field of the Invention
The present invention provides nucleotide sequences coding for ptsH and processes for the fermentative preparation of L-amino acids, particularly L-lysine, in which the ptsH gene is enhanced, using coryneform bacteria.
2. Background Information
L-amino acids, particularly L-lysine, are used in human medicine and in the pharmaceutical industry, and particularly in animal nutrition.
It is known to prepare L-amino acids by fermentation of strains of coryneform bacteria, particularly Corynebacterium glutamicum. In view of the great importance, work is constantly being carried out to improve the preparation processes. Process improvements may relate to measures involving the fermentation technique, such as, e.g., agitation and oxygen supply, or the composition of the nutrient media such as, e.g., the sugar concentration during fermentation, or the work up to the product form by, e.g., ion exchange chromatography, or the intrinsic performance properties of the microorganism itself.
In order to improve the performance properties of said microorganisms, methods of mutagenesis, selection and mutant selection are employed. Strains thereby obtained are resistant to antimetabolites such as, e.g., the lysine analogue S-(2-aminoethyl) cysteine, or auxotrophic for metabolites of regulatory importance and produce L-lysine.
For some years, methods of recombinant DNA technology have also been used to improve strains of coryneform bacteria producing L-amino acids by amplifying individual biosynthesis genes for L-amino acids and examining the effect on L-amino acid production. Review articles on this subject may be found inter alia in Kinoshita (xe2x80x9cGlutamic Acid Bacteriaxe2x80x9d, in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).
The inventors set themselves the task of providing new measures for the improved fermentative preparation of L-amino acids, particularly L-lysine.
L-amino acids, particularly L-lysine, are used in human medicine, in the pharmaceutical industry and particularly in animal nutrition. It is of general interest, therefore, to provide new improved processes for the preparation of L-amino acids, particularly L-lysine.
Where the terms L-lysine or lysine are mentioned below, they refer not only to the base but also to the salts such as, e.g., lysine monohydrochloride or lysine sulfate.
The invention provides an isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence selected from the group comprising
a) polynucleotide which is at least 70% identical to a polynucleotide coding for a polypeptide which contains the amino acid sequence of SEQ ID no. 2,
b) polynucleotide which codes for a polypeptide containing an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID no.2,
c) polynucleotide which is complementary to the polynucleotides of a) or b), and
d) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c).
The invention also provides a polynucleotide which is a DNA, preferably recombinant, which can be replicated in coryneform bacteria.
The invention also provides a polynucleotide which is an RNA.
The invention also provides a polynucleotide which is preferably a replicable DNA containing:
(i) the nucleotide sequence shown in SEQ ID no.1, or
(ii) at least one sequence which corresponds to the sequence (i) within the degeneracy region of the genetic code, or
(iii) at least one sequence which hybridises with the sequence complementary to sequence (i) or (ii), and optionally
(iv) functionally neutral sense mutations in (i).
The invention also provides
a vector containing one of the polynucleotides mentioned, and coryneform bacteria acting as host cell which contain the vector.
The invention also provides polynucleotides comprising substantially a polynucleotide sequence which may be obtained by screening by hybridising an appropriate gene bank containing the complete gene with the polynucleotide sequence corresponding to SEQ ID no. 1, with a probe which contains the sequence of the above-mentioned polynucleotide according to SEQ ID no. 1 or a fragment thereof, and isolating the DNA sequence mentioned.
Polynucleotide sequences according to the invention are suitable as hybridisation probes for RNA, cDNA and DNA, for isolating full-length cDNA which code for component H of the phosphotransferase system (ptsH) and for isolating those cDNA or genes which have great similarity of sequence with that of the gene for component H of the phosphotransferase system.
Polynucleotide sequences according to the invention are also suitable as primers for the preparation of DNA of genes which code for component H of the phosphotransferase system by the polymerase chain reaction (PCR).
The oligonucleotides acting as probes or primers contain at least 30, preferably at least 20, more particularly preferably at least 15 successive nucleotides. Oligonucleotides with a length of at least 40 or 50 nucleotides are also suitable.
xe2x80x9cIsolatedxe2x80x9d means separated from its natural surroundings.
xe2x80x9cPolynucleotidexe2x80x9d refers generally to polyribonucleotides and polydeoxyribonucleotides, which may be unmodified RNA or DNA or modified RNA or DNA.
The term xe2x80x9cpolypeptidesxe2x80x9d means peptides or proteins which contain two or more amino acids bound by way of peptide bonds.
The polypeptides according to the invention include a polypeptide according to SEQ ID no. 2, and also those with the biological activity of component H of the phosphotransferase system and also those which are at least 70% identical to the polypeptide according to SEQ ID no. 2, preferably at least 80% and in particular those which are 90% to 95% identical to the polypeptide according to SEQ ID no. 2 and have the activity mentioned.
The invention also relates to a process for the fermentative preparation of L-amino acids, particularly L-lysine, using coryneform bacteria which in particular already produce an L-amino acid and in which the nucleotide sequences coding for the ptsH gene are enhanced, particularly overexpressed.
The term xe2x80x9cenhancementxe2x80x9d describes in this context the increase in intracellular activity of one or more enzymes in a microorganism which are coded for by the corresponding DNA, by, for example, increasing the copy number of the gene or genes or alleles, using a strong promotor or using a gene or allele which codes for a corresponding enzyme with a high activity and optionally combining said measures.
The microorganisms which are the subject of the present invention may produce L-amino acids, particularly L-lysine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They may be representatives of coryneform bacteria, particularly of the Corynebacterium genus. A particular example of the Corynebacterium genus is the Corynebacterium glutamicum type which is known by experts to have the ability to produce L-amino acids.
Examples of suitable strains of the Corynebacterium genus, particularly of the Corynebacterium glutamicum type include the well known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium melassecola ATCC17965
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and L-lysine-producing mutants and strains prepared therefrom, such as, for example
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 and
Corynebacterium glutamicum DSM5715.
The inventors succeeded in isolating from C. glutamicum the new ptsH gene coding for component H of the phosphotransferase system.
In order to isolate the ptsH gene or other genes from C. glutamicum, a gene bank of this microorganism is first prepared in E. coli. The preparation of gene banks is documented in generally known textbooks and manuals. Examples include the textbook by Winnacker: Gene und Klone, Eine Einfxc3xchrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the manual by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A very well known gene bank is that of the E. coli K-12 strain W3110, which was prepared by von Kohara et al. (Cell 50, 495-508 (1987)) in xcex-vectors. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene bank of C. glutamicum ATCC13032 which was prepared using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bxc3x6rmann et al. (Molecular Microbiology 6(3), 317-326 (1992)) in turn describe a gene bank of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). In order to prepare a gene bank of C. glutamicum in E. coli, it is also possible to use plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268). Particularly suitable hosts are E. coli strains which are restriction- and recombination-defective. An example of these is the DH5xcex1MCR strain which was described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragments cloned using cosmids may then in turn be subcloned into common vectors suitable for sequencing, and then sequenced, as described in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).
The new DNA sequence coding for ptsH was obtained in this way from C. glutamicum and, as SEQ ID no. 1, forms part of the present invention. Moreover, the amino acid sequence of the corresponding protein was derived from the present DNA sequence with the methods described above. The resulting amino acid sequence of the ptsH gene product is shown in SEQ ID no. 2.
Coding DNA sequences resulting from SEQ ID No. 1 due to the degeneracy of the genetic code also form part of the invention. Experts are also familiar with conservative amino acid exchanges such as, e.g., the exchange of glycine for alanine or of aspartic acid for glutamic acid in proteins as xe2x80x9csense mutationsxe2x80x9d which do not lead to a fundamental change in the activity of the protein, i.e. which are functionally neutral. It is also known that changes at the N and/or C end of a protein do not substantially impair or may even stabilise its function. Experts may find details on this subject, inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O""Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in well known textbooks of genetics and molecular biology. Amino acid sequences which are obtained in corresponding manner from SEQ ID no. 2 and these DNA sequences encoding amino acid sequences also form part of the invention.
Similarly, DNA sequences which hybridise with SEQ ID no. 1 or parts of SEQ ID no. 1 form part of the invention. Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers obtained from SEQ ID no. 1 form part of the invention. Such oligonucleotides typically have a length of at least 15 nucleotides.
The expert may find instructions for the identification of DNA sequences by hybridisation inter alia in the manual xe2x80x9cThe DIG System Users Guide for Filter Hybridizationxe2x80x9d from Firma Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41:255-260). The expert may find instructions for the amplification of DNA sequences using the polymerase chain reaction (PCR) inter alia in the manual by Gait: Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
The inventors discovered that coryneform bacteria produce L-amino acids, particularly L-lysine, in an improved manner after overexpression of the ptsH gene.
In order to obtain overexpression, the copy number of the corresponding gene may be increased, or the promotor and regulatory region or the ribosome binding site situated upstream of the structural gene may be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same way. As a result of inducible promoters, it is also possible to increase expression in the course of fermentative L-amino acid production. Expression is also improved by measures to prolong the life of the m-RNA. Moreover, by preventing the degradation of the enzyme protein, the enzyme activity is also increased. The genes or gene constructs may either be present in plasmids with a different copy number, or integrated in the chromosome and amplified. Alternatively, overexpression of the genes concerned may be achieved by altering the composition of the medium and the way in which the culture is carried out.
The expert may find instructions on this subject inter alia in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in the European patent EPS 0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pxc3xchler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in the patent application WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in the Japanese specification JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in well known textbooks of genetics and molecular biology.
By way of example, the ptsH gene according to the invention was overexpressed using plasmids.
Suitable plasmids are those which are replicated in coryneform bacteria. Numerous well known plasmid vectors such as, e.g., pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64:549-554), pEKExc3x971 (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors such as, e.g., those based on pCG4 (U.S. Pat. No. 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (US-A 5,158,891) may be used in the same way.
Other suitable plasmid vectors include those by means of which the process of gene amplification by integration into the chromosome may be employed, as was described, e.g., by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for the duplication and amplification of the hom-thrB operon. In this method, the complete gene is cloned into a plasmid vector which is able to replicate in a host (typically E. coli), but not in C. glutamicum. Examples of suitable vectors include pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schxc3xa4fer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; U.S. Pat. No. 5,487,993), pCR(copyright)Blunt (Firma Invitrogen, Groningen, Niederlande; Bernard et al., Journal of Molecular Biology, 234:534-541 (1993)) or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516). The plasmid vector which contains the gene to be amplified is then transferred by conjugation or transformation into the desired strain of C. glutamicum. The conjugation method is described, for example, in Schxc3xa4fer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods of transformation are described, for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination using a xe2x80x9ccross overxe2x80x9d event, the resulting strain contains at least two copies of the gene concerned.
The invention also provides, therefore, a process for the fermentative preparation of L-amino acids, particularly L-lysine, wherein a strain transformed with a plasmid vector is used and the plasmid vector carries the nucleotide sequence of the gene coding for component H of the phosphotransferase system.
Moreover, it was found that by exchanging the amino acid L-alanine in position 25 of the protein component H of the phosphotransferase system (see SEQ ID NO:2) for any other proteinogenic amino acid, particularly L-threonine (see SEQ ID NO:4), with the exception of L-alanine, enhancement takes place and coryneform bacteria which bear the corresponding amino acid exchange produce L-lysine in an improved manner. The exchange of L-alanine for L-threonine in position 25 of the amino acid sequence may be carried out preferably by exchanging the nucleobase adenine in position 235 for guanine, as shown in the nucleotide sequence according to SEQ ID NO:3.
Conventional mutagenesis methods using mutagenic substances such as, for example, N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine or ultraviolet light may be used for mutagenesis. Moreover, in vitro methods such as, for example, a treatment with hydroxylamine or mutagenic oligonucleotides or the polymerase chain reaction (PCR) may be used for mutagenesis.
Accordingly, the invention also provides coryneform bacteria which contain a protein component H of the phosphotransferase system in which the amino acid sequence in position 25 shown under SEQ ID NO:2 is exchanged for another amino acid except L-alanine. A further aspect of this invention is coryneform bacteria which contain a corresponding protein in which the amino acid L-alanine in position 25 of the protein (see SEQ ID NO:2) is exchanged for L-threonine (see SEQ ID NO:4).
Accordingly, the invention also provides polynucleotide sequences originating from coryneform bacteria which contain genes or alleles which encode the above-mentioned protein components H of the phosphotransferase system.
The present invention also provides coryneform bacteria which contain a DNA encoding a protein component H of the phosphotransferase system which is characterised by the exchange of L-alanine for L-threonine in position 25, which DNA contains adenine instead of the nucleobase guanine in position 235 (see SEQ ID NO:1), as shown in SEQ ID NO:3.
In addition, it may be advantageous for the preparation of L-amino acids, particularly L-lysine, to enhance not only the ptsH gene but also other genes of the biosynthesis pathway of the desired L-amino acid so that one or more enzymes of the biosynthesis pathway in question, glycolysis, anaplerotic reactions or amino acid export, is overexpressed.
For the preparation of L-lysine, for example, it is possible to overexpress simultaneously one or more of the genes selected from the group comprising
the dapA gene coding for dihydrodipicolinate synthase (EP-B 0 197 335),
the gap gene coding for glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
the tpi gene coding for triosephosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
the pgk gene coding for 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
the ptsM gene coding for component M of the phosphoenolpyruvate-sugar-phosphotransferase system (ptsM) (Lee et al. (1994), FEMS Microbiology Letters 1-2, 137-145),
the pyc gene coding for pyruvate carboxylase (DE-A-198 31 609), and
the lysE gene coding for lysine export (DE-A-195 48 222).
Moreover, for the production of L-amino acids, particularly L-lysine, it may be advantageous, in addition to the ptsH gene, simultaneously to attenuate
the pck gene coding for phosphoenolpyruvate carboxykinase (DE 199 50 409.1, DSM 13047) and/or
the pgi gene coding for glucose-6-phosphate isomerase (U.S. Pat. No. 09/396,478, DSM 12969)
the poxB gene coding for pyruvate oxidase (DE 19846499.1; DSM 13114).
Moreover, for the production of L-amino acids, particularly L-lysine, it may be advantageous, in addition to the overexpression of the ptsH gene, to exclude unwanted side reactions (Nakayama: xe2x80x9cBreeding of Amino Acid Producing Micro-organismsxe2x80x9d, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
The microorganisms produced according to the invention may be cultivated continuously or batchwise in the batch process (batch cultivation) or in the fed-batch or repeated fed-batch process in order to produce L-amino acids, particularly L-lysine. Summaries of well known cultivation methods are described in the textbook by Chmiel (Bioprozesstechnik 1. Einfxc3xchrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must satisfy the requirements of the strains concerned in a suitable manner. Descriptions of culture media of various microorganisms are contained in the manual xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d of the American Society for Bacteriology (Washington D.C., USA, 1981). Suitable sources of carbon include sugars and carbohydrates such as, e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as, e.g., soyabean oil, sunflower oil, groundnut oil and coconut fat, fatty acids such as, e.g., palmitic acid, stearic acid and linoleic acid, alcohols such as, e.g., glycerol and ethanol and organic acids such as, e.g., acetic acid. Said substances may be used individually or as mixtures. Suitable sources of nitrogen include organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, maize swelling water, soyabean flour and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The sources of nitrogen may be used individually or as a mixture. Suitable sources of phosphorus include phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium must also contain salts of metals such as, e.g., magnesium sulfate or iron sulfate which are necessary for growth. Finally, essential growth-promotors such as amino acids and vitamins may be used in addition to the substances mentioned above. Moreover, suitable preliminary stages may be added to the culture medium. The substances used may be added to the culture in the form of a single preparation or fed in a suitable manner during cultivation.
In order to control the pH of the culture, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammoniacal gas liquor or acid compounds such as phosphoric acid or sulfuric acid may be used in a suitable manner. Antifoaming agents such as, e.g., fatty acid polyglycol esters may be used to control foam development. In order to maintain the stability of plasmids, suitable selectively acting substances such as, e.g., antibiotics may be added to the medium. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures such as, e.g., air may be introduced into the culture. The temperature of the culture is normally from 20xc2x0 C. to 45xc2x0 C. and preferably from 25xc2x0 C. to 40xc2x0 C. The culture is continued until an L-lysine maximum has formed. This objective is normally achieved within 10 hours to 160 hours.
The invention also provides, therefore, a process for the fermentative preparation of L-amino acids, particularly L-lysine, wherein the following steps are carried out:
a) Fermentation of coryneform bacteria producing L-amino acids in which at least the ptsH gene coding for component H of the phosphotransferase system is enhanced, particularly overexpressed.
b) Enrichment of the L-amino acid in the medium or in the cells of the bacteria, and
c) Isolation of the L-amino acid.
The analysis of L-lysine may be carried out by anion exchange chromatography followed by ninhydrin derivatisation, as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190).
The process according to the invention is used for the fermentative preparation of L-amino acids, particularly L-lysine.